Abstract
Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.
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Abbreviations
- AF:
-
Actin filament
- BSA:
-
Bovine serum albumin
- BY-2 cells:
-
Tobacco Bright Yellow-2 cells
- DMSO:
-
Dimethyl sulfoxide
- GFP:
-
Green fluorescent protein
- MBD:
-
Microtubule binding domain of the microtubule associated protein 4
- MBS ester:
-
3-Maleimidobenzoyl-N-hydroxysuccinimide ester
- MSB:
-
Microtubule stabilizing buffer
- MT:
-
Microtubule
- PFA:
-
Paraformaldehyde
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Acknowledgements
The participation of M. Szechyńska-Hebda, E. Dubas and M. Wędzony was supported by the 5th Framework Programme project CROPSTRESS contract number QLK5-CT-2002-30424.
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Szechyńska-Hebda, M., Wędzony, M., Dubas, E. et al. Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol. Plant Cell Rep 25, 758–766 (2006). https://doi.org/10.1007/s00299-005-0089-y
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DOI: https://doi.org/10.1007/s00299-005-0089-y